title: Resistance of human cytomegalovirus to cyclopropavir maps to a base pair deletion in the open reading frame of UL97.
abstract: Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) than the wild-type virus (EC50 = 0.36 ± 0.11 μM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.
authors.affiliation: Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences, Drake University, Des Moines, Iowa, USA. brian.gentry@drake.edu
authors.forename: Brian G
authors.initials: BG
authors.lastname: Gentry
authors.forename: Laura E
authors.initials: LE
authors.lastname: Vollmer
authors.forename: Ellie D
authors.initials: ED
authors.lastname: Hall
authors.forename: Katherine Z
authors.initials: KZ
authors.lastname: Borysko
authors.forename: Jiri
authors.initials: J
authors.lastname: Zemlicka
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Kamil
authors.forename: John C
authors.initials: JC
authors.lastname: Drach
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003521:Cyclopropanes / Q000494:pharmacology; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000821:virology; D016368:Frameshift Mutation; D015774:Ganciclovir / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D003521:Cyclopropanes; D047428:Protein Kinase Inhibitors; D014764:Viral Proteins; D006147:Guanine; C495219:cyclopropavir; D011494:Protein Kinases; D015774:Ganciclovir